人血管能抑素基因的克隆、表达及其表达产物的纯化和生物活性测定  

Cloning and Expression of Human Canstatin and its Purification and Bioactivity

作　　者：贺国安[1] 罗进贤[1] 张添元[1] 胡志上[1] 李瑞芳[1] 

机构地区：[1]中山大学基因工程教育部重点实验室,广州510275

出　　处：《微生物学报》2003年第5期607-612,共6页Acta Microbiologica Sinica

基　　金：广东省重点科技项目 (2KM0 2 5 0 2G)~~

摘　　要：以人胎盘脐带组织为材料 ,提取组织总RNA ,用net RT PCR方法合成人血管能抑素cDNA基因 ,将该cDNA克隆进pSP72载体获得重组质粒pSP72C ,DNA序列分析结果与预期序列一致。用BamHⅠ和NdeⅠ双酶切 ,切下pSP72C上的血管能抑素cDNA ,插入pET 3c载体的相应位点获得重组表达质粒pETC ,转化E .coliBL2 1 (DE3 ) ,SDS PAGE分析显示 :在IPTG诱导下 ,血管能抑素基因获得了高效表达 ,表达量约占菌体总蛋白的 2 7 9% ,主要以包涵体形式存在。包涵体经过洗涤、裂解、蛋白复性以及SephadexG 75凝胶过滤层析等步骤后 ,获得了纯度达 91 4%的人血管能抑素。CAM实验证明 1 0 μg纯化蛋白就能显著抑制鸡胚新生血管生成。Total RNA was extracted from placenta umbilical tissue and the cansta tin cDNA was amplified from total RNA by net-RT-PCR technique. The amplified c DN A was cloned into pSP72 and sequenced. The canstatin cDNA was cut down from pSP7 2C with BamHⅠ/NdeⅠ and ligated into the vector pET-3c. The resulta nt plasmid p ETC was then transformed into E coli BL21(DE3). The canstatin gene was ef ficie ntly expressed after IPTG induction as a 25 kD band on SDS-PAGE. The expressed p roduct constituted approximately 27 9 % of the total bacterial proteins estimat e d by densitometry and existed mainly as inclusion body. The inclusion bodies wer e washed, lysed, refolded and purified on the Sephadex G-75 gel filtration colu m n to a purity of 91 4 %. CAM assay showed that 10 μg purified canstatin is eno ugh to inhibit the angiogenesis of chichen embryo microcapillary vessel.

关 键 词：血管能抑素基因 克隆 表达 蛋白纯化 抗血管生成 肿瘤 

分 类 号：Q78[生物学—分子生物学] R730[医药卫生—肿瘤]